Excitotoxicity is related to excessive activation of glutamate receptors which results in neuronal cell death. The physiological function of glutamate receptors is the mediation of ligand-gated cation channels with the concomitant influx of calcium, sodium and potassium through this receptor-gated channel. The influx of these cations is essential for maintaining membrane potentials and the plasticity of neurons which in itself plays a pivotal role in cognitive function of the central nervous system. Li, H. B., et al., Behav. Brain Res., 83:225-228 (1997); Roesler, R., et al., Neurology, 50:1195 (1998); Wheal, H. V., et al., Prog. Neurobiol., 55:611-640 (1998); Wangen, K., et al., Brain Res., 99:126-130 (1997). Excitotoxicity plays an important role in neuronal cell death following acute insults such as hypoxia, ischemia, stroke and trauna, and it also plays a significant role in neuronal loss in AIDS dementia, epilepsy, focal ischemia. Coyle, J. T. and Puttfarken, P., Science, 262:689-695 (1993). Neurodegenerative disorders, such as Huntington""s disease (HD), Alzheimer""s disease (AD), Parkinson""s disease (PD) and amyotrophic lateral sclerosis (ALS), are characterized by the progressive loss of a specific population of neurons in the central nervous system. Growing evidence suggests that glutamate-mediated excitotoxicity may be a common pathway which contributes to neuronal cell death in a wide range of neurological disorders. Coyle, J. T. and Puttfarken, P., Science, 262:689-695 (1993).
The molecular mechanisms of excitotoxicity-mediated neuronal cell death remains obscure. Over-production of free radicals that lead to impairment of mitochondrial function is the most widely held hypothesis. Beal, M. F., et al., Ann. Neurol., 38:357-366 (1995); Coyle, J. T. and Puttfarken, P., Science, 262:689-695 (1993) However, it is unclear whether the increase of free radicals is the precursor that initiates neuronal degeneration or, rather, a subsequent consequence of neuronal degeneration. Interestingly, administration of antioxidants has little neuroprotective effect in patients suffering from various neurodegenerative diseases. Shults, C. W., et al., Neurology, 50:793-795 (1998). Thus, some other mechanism(s) must exist for excitotoxicity-induced neuronal cell death.
c-Jun N-terminal kinases (JNKs) are identified as kinases which are activated upon stimulation by various environmental stimuli such as UV light, xcex irradiation and mitogenic signals. Hibi, M., et al., Genes Dev., 7:2135-2148 (1993); Kyriakis, J. M., et al., Nature, 369:156-160 (1994). The precise biological function of JNKs remains to be explored. However, some recent reports suggest that JNKs are involved in neuronal apoptosis induced by deprivation of survival factors, i.e., neurotrophic factors which support neuronal survival. Ham, J., et al., Neuron, 14:927-939 (1995).
Mixed-lineage kinases (MLKs), so called because these proteins contain structural domains associated with a variety of cell types, were cloned from a cDNA library derived from mRNA from cancer tissue. MLKs were initially thought to participate in the oncogenesis of some cancers, although high levels of expression of MLKs were found in the normal brain. Dorow, D. S., et al., Eur. J. Biochem., 213:701-710 (1993); Dorow, D. S., et al., Eur. J. Biochem., 234:492-500 (1995).
Searching for biochemical targets which are amenable to screening for neuroprotective therapeutic agents is of central concern in neuroscience today. However, no clinically available pharmaceutical tool to date is employed for blocking excitotoxicity and preventing neuronal cell loss in various neurological disorders due to a lack of suitable biochemical targets. Glutamate receptor antagonists, such as MK-801, although successful in protecting neurons in animal experiments, have all failed in the clinical setting due to their blockage of cognitive function mediated by the receptors, as well as high toxicity to the central nervous system. Thus, an understanding of the molecular mechanism(s) of neuronal cell death induced by excitotoxicity is essential for the identification of new biochemical targets and the establishment of reliable methods for screening new therapeutic drugs from chemical libraries that can be utilized in the treatment of a variety of neurological disorders.
This invention relates to the discovery that inhibiting a JNK or MLK within a hippocampal neuronal cell can protect the cell from apoptosis. As such, JNK and MLK can be used as drug targets to screen for therapeutic agents to prevent glutamate or kainic acid mediated toxicity, to block excitotoxicity and to prevent neuronal loss in a variety of neurological conditions, such as Huntington""s disease and Alzheimer""s disease.
In one aspect of the invention, a method is described for assessing a compound""s ability to inhibit neuronal cell death, and thus to identify compounds that can be used to prevent and/or treat neurological conditions. According to the method, neuronal cells having activated MLK and/or JNK activity are contacted with a compound and the number of neuronal cells that die is determined. A decrease in the number of dead neuronal cells in the presence of the compound compared to the number of dead neuronal cells in the absence of the compound is indicative of the compound""s ability to inhibit neuronal cell death. Preferably, the neuronal cells are apoptotic neurons (i.e., cell death caused by a neurological condition) or neurons that are induced to undergo apoptosis, such as by contacting the neuronal cells with neurotoxin (e.g., glutamate, quinolinic acid or kainic acid); or by genetic manipulation of the neuronal cells. Most preferred are HN33 hippocampal neuronal cells.
In another embodiment, the invention features a method for testing a compound""s potential as a drug for treating a mammal (e.g., a human) susceptible to or having a neurological condition by (1) contacting a compound with a JNK (e.g., JNK3) or MLK (e.g., MLK2); (2) measuring the level of a JNK-associated or MLK-associated activity (e.g., a kinase activity); and (3) comparing the level of the JNK-associated or MLK-associated activity in the presence of the compound with the level of the JNK-associated or MLK-associated activity in the absence of the compound. The compound is a potentially useful drug for treating the mammal when the level of the JNK-associated or MLK-associated activity in the presence of the compound is less than the level of the JNK-associated or MLK-associated activity in the absence of the compound.
The JNK or MLK can be within a cell, which can be an animal (e.g., human) cell in vivo. When the JNK or MLK is within a cell, the JNK-associated or MLK-associated activity can be apoptosis, which can be measured by a TUNEL assay (described below). Apoptosis within such a cell can be induced by introducing into the cell a huntingtin protein that has at least 40 consecutive glutarnic acids (e.g., polyglutamine stretch-expanded huntingtin). Alternatively, apoptosis can be induced by introducing into the cell the C-terminal 100 amino acids of an amyloid precursor protein (APP). Preferably, the huntingtin protein or the amyloid precursor protein is introduced by a vector, especially a nucleic acid vector. When the cell is within an animal, the JNK-associated or MLK-associated activity can be neurodegeneration.
The invention also features a method for testing a compound""s potential as a drug for treating a mammal (e.g., a human) susceptible to or having a neurological condition by (1) contacting a compound with a neuronal cell containing a JNK (e.g., JNK3) or MLK (e.g., MLK2); (2) measuring the level of a JNK or MLK protein activity (e.g., kinase activity, such as the presence or amount of phosphorylated product) in the cell; and (3) comparing the level of the INK or MLK protein activity in the cell in the presence of the compound with the level of the JNK or MLK protein activity in the cell in the absence of the compound. The compound is a potentially useful drug for treating the mammal when the level of the JNK or MLK protein activity in the cell in the presence of the compound is less than the level of the JNK or MLK protein activity in the cell in the absence of the compound. Alternatively, cell viability can be ascertained by determining the degree of neuronal cell death, wherein a decreased number of dead neuronal cells in the presence of the compound compared to the number of dead neuronal cells in the absence of the compound is indicative of the compound""s ability to inhibit JNK or MLK protein activity, thereby preventing neuronal cell death.
In another aspect, the invention provides a method for testing the potential of a JNK or MLK inhibitor as a drug for treating a mammal (e.g., a human) susceptible to or having a neurological condition. The method can bc performed on compounds identified as JNK and/or MLK inhibitory agents using the methods of this invention to confirm their inhibitory effectiveness under apoptotic conditions. Accordingly, the method provides (1) incubating a neuronal cell in the presence of a JNK or MLK inhibitor; (2) contacting surviving cells with an agent that induces apoptosis in the cell; and (3) comparing the occurrence of apoptosis in the cell in the presence of the JNK or MLK inhibitor with the occurrence of apoptosis in the cell in the absence of the JNK or MLK inhibitor. The compound is a potentially useful drug for treating the mammal when the occurrence of apoptosis in the cell in the presence of the JNK or MLK inhibitor is less than the occurrence of apoptosis in the cell in the absence of the JNK or MLK inhibitor.
The methods of the invention are used to identify inhibitors of JNK or MLK which are potentially useful for the treatment of a neurological condition, including neuronal cell death following acute insults such as hypoxia, ischemia, stroke, and trauma. Other neurological conditions treatable with compounds identified by the methods of the invention include AIDS dementia, epilepsy, focal ischemia, Huntington""s disease, Alzheimer""s disease, Parkinson""s disease, and amyotrophic lateral sclerosis. Each of these conditions are characterized by the progressive loss of a specific population of neurons in the central nervous system. The methods of the invention are particularly useful in finding compounds which can be used to prevent and/or treat neurological conditions, including genetic neurological conditions. The invention also pertains to compounds, identified using the methods described herein, that inhibit MLK and/or JNK activity and that prevent neuronal cell death occurring in a mammal susceptible to or having a neurological condition, particularly neurodegenerative diseases, such as Huntington""s disease and Alzheimer""s disease.
The invention also provides methods for preventing and/or treating neuronal conditions in a mammal comprising administering to a mammal, in need thereof, an effective therapeutic amount of a compound that inhibits JNK and/or MLK. The inhibitory effects of the compound will reduce and/or prevent neuron cell death occurring in a mammal susceptible to or having a neurological condition. In a preferred embodiment, the neurological condition is a neurological disease whereby glutamate or kainic acid mediated excitotoxicity is involved in neuronal cell death. JNK and/or MLK inhibitors identified using any of the methods described herein are useful as therapeutic or prophylatic drugs to prevent neuronal loss.